Abstract
Purpose: :
The depolarizing light-evoked responses of ON bipolar cells (ON-BCs) are generated by a metabotropic signaling cascade involving the glutamate receptor, mGluR6, the heterotrimeric G protein, Gαo, and the cationic channel, TRPM1. The deactivation of Gαo is required to generate the rising phase of the light-evoked response in ON-BCs. Fast kinetics of the ON-BC response suggest that slow spontaneous deactivation of Gαo is catalyzed by GTPase Activating Proteins (GAP). However, the identity of the dominant GAP in the ON-BC that drives generation of light responses is not known. Here we studied the role of RGS7 and RGS11 proteins in ON-BC function.
Methods: :
We generated a line of knockout mice with complete elimination of RGS7. These mice were crossed with RGS11 knockouts to generate a line of double knockouts (DKO). Protein expression, localization, and synaptic morphology were analyzed by Western blotting, immunohistochemistry and electron microscopy, respectively. The light-evoked responses of ON-BCs were studied in intact retinas by ERG, and in single rods and rod bipolar cells from dark adapted retina using suction and patch electrodes, respectively.
Results: :
We found that elimination of RGS7 alone did not alter the light-evoked responses of ON-BCs. However, simultaneous knockout of RGS7 and RGS11 resulted in severe disruption of ON-BC function with no effect on rod photoresponses. ERG recordings showed that threshold for the generation of b waves was much higher in the DKO mice. Furthermore, the onset of the b-wave was slowed by more than an order of magnitude. Single cell recordings revealed that flash responses were completely absent in DKO rod ON-BCs. However, bright steps of light in DKO rod ON-BCs elicited inward currents that were ~50-fold smaller and had ~20-fold slower onset as compared to wild-type rod ON-BCs. The synaptic morphology and retina cytoarchitecture in DKO was normal.
Conclusions: :
These results are consistent with persistently high G protein activity in the dendrites of DKO ON-BCs, suggesting that RGS7 and RGS11 together provide the dominant GAP in the mGluR6 signaling cascade that is essential for setting the sensitivity and temporal resolution of ON-BCs.
Keywords: bipolar cells • retina: distal (photoreceptors, horizontal cells, bipolar cells) • signal transduction